We have developed methods to measure the hypothalamic hormone, thyrotropin releasing hormone by radioimmunoassay and bioassay techniques. In crude hypothalamic extracts there is excess immunologic to biologic activity of TRH caused by variable amounts of inhibitory substances such as somatostatin. The immunologic to biologic ratio is lower during cold stress but not in hyperthyroidism or hypothyroidism. In purified extracts there is a constant amount of TRH under all conditions. Thus TSH secretion is primarily regulated by hypothalamic inhibitory factors.